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首页> 外文期刊>Molecular and Cellular Biology >Extracellular Signal-Regulated Kinases Phosphorylate Mitogen-Activated Protein Kinase Phosphatase 3/DUSP6 at Serines 159 and 197, Two Sites Critical for Its Proteasomal Degradation
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Extracellular Signal-Regulated Kinases Phosphorylate Mitogen-Activated Protein Kinase Phosphatase 3/DUSP6 at Serines 159 and 197, Two Sites Critical for Its Proteasomal Degradation

机译：胞外信号调节激酶磷酸化丝裂原活化的蛋白激酶磷酸酶3 / DUSP6在丝氨酸159和197，这两个部位对于其蛋白酶体降解至关重要

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Mitogen-activated protein (MAP) kinase phosphatases (MKPs) are dual-specificity phosphatases that dephosphorylate phosphothreonine and phosphotyrosine residues within MAP kinases. Here, we describe a novel posttranslational mechanism for regulating MKP-3/Pyst1/DUSP6, a member of the MKP family that is highly specific for extracellular signal-regulated kinase 1 and 2 (ERK1/2) inactivation. Using a fibroblast model in which the expression of either MKP-3 or a more stable MKP-3-green fluorescent protein (GFP) chimera was induced by tetracycline, we found that serum induces the phosphorylation of MKP-3 and its subsequent degradation by the proteasome in a MEK1 and MEK2 (MEK1/2)-ERK1/2-dependent manner. In vitro phosphorylation assays using glutathione S-transferase (GST)-MKP-3 fusion proteins indicated that ERK2 could phosphorylate MKP-3 on serines 159 and 197. Tetracycline-inducible cell clones expressing either single or double serine mutants of MKP-3 or MKP-3-GFP confirmed that these two sites are targeted by the MEK1/2-ERK1/2 module in vivo. Double serine mutants of MKP-3 or MKP-3-GFP were more efficiently protected from degradation than single mutants or wild-type MKP-3, indicating that phosphorylation of either serine by ERK1/2 enhances proteasomal degradation of MKP-3. Hence, double mutation caused a threefold increase in the half-life of MKP-3. Finally, we show that the phosphorylation of MKP-3 has no effect on its catalytic activity. Thus, ERK1/2 exert a positive feedback loop on their own activity by promoting the degradation of MKP-3, one of their major inactivators in the cytosol, a situation opposite to that described for the nuclear phosphatase MKP-1.
机译：丝裂原活化蛋白（MAP）激酶磷酸酶（MKP）是双特异性磷酸酶，可将MAP激酶中的磷酸苏氨酸和磷酸酪氨酸残基脱磷酸化。在这里，我们描述了一种新型的翻译后机制，用于调节MKP-3 / Pyst1 / DUSP6（MKP家族的成员），该成员对细胞外信号调节激酶1和2（ERK1 / 2）失活具有高度特异性。使用成纤维细胞模型，其中四环素诱导MKP-3或更稳定的MKP-3-绿色荧光蛋白（GFP）嵌合体的表达，我们发现血清诱导MKP-3的磷酸化及其随后的降解。蛋白酶体依赖于MEK1和MEK2（MEK1 / 2）-ERK1 / 2。使用谷胱甘肽 S -转移酶（GST）-MKP-3融合蛋白的体外磷酸化分析表明，ERK2可以使159和197丝氨酸上的MKP-3磷酸化。四环素诱导的细胞克隆表达单丝氨酸或双丝氨酸MKP-3或MKP-3-GFP突变体证实这两个位点在体内被MEK1 / 2-ERK1 / 2模块靶向。 MKP-3或MKP-3-GFP的双丝氨酸突变体比单突变体或野生型MKP-3更有效地防止降解，表明ERK1 / 2丝氨酸磷酸化可增强MKP-3的蛋白酶体降解。因此，双重突变导致MKP-3的半衰期延长了三倍。最后，我们表明，MKP-3的磷酸化对其催化活性没有影响。因此，ERK1 / 2通过促进MKP-3的降解对其自身的活性产生正反馈回路，MKP-3是其在细胞质中的主要灭活剂之一，与核磷酸酶MKP-1所描述的情况相反。

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